Technical Field
The present invention is related to the pharmaceutical field. More specifically, the present invention relates to: Hsp70 peptidic binders as anti-tumor therapeutic agents, the use thereof, as well as a pharmaceutical composition containing the same, optionally in combination with other chemotherapeutic agents; to a gene construct for the expression of said binder; to production processes; to a process for the evaluation of tumor cells; and to a method of sensitization of tumors.
Prior Art
In 1962, Ritossa and collaborators, when submitting Drosophila melanogaster larvae to temperatures 10 degrees Celsius higher than their usual temperature, observed the activation of specific genes. This treatment was referred to as heat shock and, in 1974, the products of these genes were identified as Heat Shock Proteins (Hsps). Subsequent work demonstrated that these proteins can be induced in all living beings when cells respond to high temperatures and other manifestations of stress by the rapid synthesis of Hsps, being, therefore, proteins having a high degree of phylogenetic conservation.
Hsps are categorized into several families, named according to their molecular weight, thus, Hsp70 means a Hsp protein having 70 kilodaltons (kD), for example. These proteins are produced/induced when cellular stresses, such as high temperatures, disturb the physiology of the organism. The main agents that induce the production of Hsps are temperature elevation, oxidative stress, nutritional deficiency, ultraviolet radiation, chemical agents, viral infections, ischemia and reperfusion damage, exposure to pro-inflammatory mediators and treatment with non-steroidal drugs. Said proteins can be located in different cellular compartments, so that, in normal cell conditions, Hsp70 is located in the endoplasmic reticulum lumen, in addition to the cytoplasm and the nucleus.
Over the course of evolution, the proteins of the Hsp70 family such as, for example, the HspBP1 protein, maintain similarity of structure and sequence. However, between human and bacterial Hsp70, there is approximately 50% of conservation. The idea that human Hsp70 and their bacterial counterparts have the same properties is not yet established. However, they do have in common an atpasic N-terminal domain and a C-terminal domain that is able to bind to peptides. This is a relevant distinction between the present invention and the patent and non-patent references that circumscribe the topic.
The HspBP1 protein is deemed to be a member of a family of proteins present in eukaryotic cells, identified as HSP 70 nucleotide exchange factors, acts as co-chaperone, which exhibit varying degrees of enclosure and a specificity of the species. The HspBP1 protein is located mainly in the cytoplasm and in the nucleus, but can also be found outside the cell. Said protein, mostly expressed in the heart and skeletal muscle, is capable of binding to Hsp70 protein, inhibiting its activity and promoting dissociation of nucleotides within the ATPase domain. In addition, the HspBP1 protein inhibits the activity of HspA1A chaperone, changing the conformation of the ATP binding domain of HspA1A and obstructing the ATP bond. The HspBP1 protein has a role in the modulation of chaperones located on the surface of tumor cells.
There are Hsps that are constitutively produced and Hsps induced under stress. The main function of constitutive Hsps is probably acting as chaperone for proteins nascent during protein synthesis, having the function of, through the recognition and connection to these polypeptides, promoting and ensuring its correct folding, which is essential for its correct operation. Subsequent studies verified an interaction between Hsp70 and HspBP1, showing that HspBP1 binds to Hsp70, modulating it. In such case, Hsp70 assumes the role as chaperone and HspBP1 as co-chaperone, i.e., binds to the chaperone, modulating it.
The function of induced Hsps seems to be central to the maintenance of cellular homeostasis, by binding themselves to total or partially denatured proteins, helping them to recover their original tertiary structure.
The patent literature and other documents from the prior art also portray studies involving HspBP1 protein.
Patent application WO 2001/81545 shows a method for the modulation of the expression of extracellular genetic material through the use of HspBP1 polypeptides and polynucleotides. The present invention differs from the above document because, among other technical reasons, unlike the present invention, it does not describe a method of genetic material expression modulation through the modulating activities of protein HspBP1. Nor the use of HspBP1 protein non-natural fragment(s) for such applications is revealed or suggested.
The article “Increased Expression of Hsp70 Cochaperone HspBP1 in Tumors”, published in Oct. 27, 2003 in Tumour Biology: the Journal of the International Society for Oncodevelopmental Biology and Medicine, reports the alteration of HspBP1 protein levels in murine tumors. Said Article provides that tumors in mouse have high levels of HspBP1 protein compared to normal tissues. This study also describes that the HspBP1 protein binds to and reduces the activity of the Hsp70 protein. The present invention differs from the above Article, because, among other technical reasons, it surprisingly lists the connection between HspBP1 to Hsp70 and reduction of tumor growth. With this, it suggests the direct administration of the HspBP1 protein for therapeutic purposes, which was not disclosed or even suggested in said Article. Nor the use of HspBP1 protein non-natural fragment(s) for such applications is revealed or suggested.
The article “HspBP1 levels are elevated in breast tumor tissue and inversely related to tumor aggressiveness”, published in Nov. 6, 2008 in Cell Stress and Chaperones, discloses that HspBP1 protein is increased in tumor tissues in relation to the normal adjacent tissues. Said article also describes that the HspBP1 co-chaperone levels are inversely related to the aggressiveness of the tumor. The article does not anticipate or even suggest the object of the present invention, which is characterized by providing an anti-tumor therapeutic option through the direct administration of HspBP1 protein. Nor the use of HspBP1 protein non-natural fragment(s) for such applications is revealed or suggested.
The article “The Heat Shock-binding Protein (HspBP1) Protects Cells against the Cytotoxic Action of the Tag7-Hsp70 Complex”, published on Mar. 25, 2011 in The Journal of biological chemistry, discloses the inhibitory effect of HspBP1 protein on the Hsp70-Tag7 complex cytotoxic activity (Tag7 being a peptidoglycan recognition protein PGRP-S), which form a cytotoxic complex against certain tumor cells. According to the article, such inhibitory effect can be a defense mechanism of normal cells of adjacent tissues against the cytotoxicity of said complex. The present invention differs from that document because, among other technical reasons, it describes the administration of HspBP1 protein in order to decrease tumor growth. Nor the use of HspBP1 protein non-natural fragment(s) for such applications is revealed or suggested.
The article “Heat shock protein 70-binding protein 1 is highly expressed in high-grade gliomas, interacts with multiple heat shock protein 70 family members, and specifically binds brain tumor cell surfaces”, published on Jul. 30, 2009 in Cancer Science, considered the closest document in relation to the present invention, discloses that HspBP1 protein binds to the Hsp70 proteins located at the surface of tumor cells, internalizing itself therein and playing a role in chaperone modulation. The present invention differs substantially from said article because, among other differences, it proves that through the administration of HspBP1 protein, a tumor growth reduction is achieved. Nor the use of HspBP1 protein non-natural fragment(s) for such applications is revealed or suggested in such prior art.
The article by Tanimura et al. “Anticancer Drugs Up-regulate HspBP1 and thereby antagonize the Prosurvival Function of Hsp70 in Tumor Cells” (The Journal of Biological Chemistry vol. 282, No. 49, pp. 35430-35439, Dec. 7, 2007) describes that the increased HspBP1 expression antagonizes the Hsp70 pro-survival effect, sensitizing tumor cells to cathepsin-mediated death. However, they have not demonstrated this action in B16F10 cells. Nor the use of HspBP1 protein non-natural fragment(s) for such applications is revealed or suggested.
In view of the documents listed above, it will be observed that in the literature, no documents were found relating the use of HspBP1 protein, or non-natural fragment(s) thereof, in the preparation of medicinal products for the reduction of tumor growth through direct administration, nor suggesting that their results would be surprisingly as better as they are in this invention.
Currently, the treatment regimens for patients with tumors are performed by combining different chemotherapeutic drugs, most of them having low specificity and high cytotoxicity. The chemotherapeutic drugs have their therapeutic action based essentially on injury to cell mitosis, effectively affecting fast growing cells. Therefore, they cause cell damage, being called cytotoxic or cytostatic. Some of these drugs cause apoptosis (programmed cell death), causing side effects in other rapid division cells of the body that are not necessarily linked to the disease, such as, for example, intestinal epithelium cells and cells responsible for hair growth.
Another disadvantage of the current chemotherapy therapeutic regimen is the fact that during the treatment period, neoplastic cells become resistant to current chemotherapeutic drugs. One recent aspect is the development of a mechanism of pumps that are located on the surface of cancerous cells, which active and continuously move chemotherapeutic agents out of the target cell. This fact is connected, mainly, to the P-glycoprotein, which currently has become an important target of studies.
HspBP1 and Hsp70 proteins can be secreted from tumor cells and it is well-established that Hsp70 is present in human serum. Among other results, the present inventors have demonstrated that patients with malignant breast tumors had twice the level of HspBP1 in their sera in comparison with the control patients and malignant breast tumors contained HspBP1 (and Hsp70) levels 4 times higher than in normal adjacent tissue. In these studies, it was also observed that the tumors having the lowest HspBP1 levels became metastatic and resulted in more deaths among the patients. A recent analysis showed that extracellular HspBP1 co-chaperone binds to the surface of tumor cells by binding to Hsp70 chaperone (which, in turn, can be found in the surface of tumor cells), causing tumor reduction effects. These results support the inventors' hypothesis that the HspBP1 co-chaperone protein, or non-natural fragment(s) thereof, reduces tumor growth through its binding to cell surface proteins of tumors.
The present invention differs and surpasses the current chemotherapy therapeutic regimen as it is proposed as a highly specific and negligibly cytotoxic alternative for tumor growth reduction, since it comprises a Hsp70 binding compound (protein which is present in the surface of tumor cells), negatively modulating it, which results in anti-tumor effect. In addition, because it is specific, the approach of the present invention does not have the disadvantages related to drugs currently used in the current chemotherapy therapeutic regimen.